Compositions and methods for increasing protein half-life in a serum

ABSTRACT

Novel antibodies, such as single domain antibodies (sdAbs), or fragments thereof that specifically bind a transferrin are described. Compositions, methods and systems for increasing the half-life of a target protein in a serum using an antibody or fragment thereof against a transferrin are described.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.14/903,156, filed Jan. 6, 2016, which is a Section 371 of InternationalApplication No. PCT/US14/45768, filed Jul. 8, 2014, which was publishedJan. 15, 2015 under International Publication No. WO 2015/006337 A2,which claims priority to U.S. Provisional Patent Application No.61/843,628, filed Jul. 8, 2013, the discloses of which are incorporatedherein by reference.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “689296-2U2 Sequence Listing,” creation date of May 14, 2019,and having a size of 40.9 kb. The sequence listing submitted via EFS-Webis part of the specification and is herein incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

Pharmacokinetics of a drug candidate is a critical parameter and oftenlargely determines whether or not the drug candidate will be furtherdeveloped into a drug or used for a therapeutic application. Inparticular, pharmacokinetic studies of protein- and peptide-basedtherapeutics, including antibodies, have demonstrated that suchtherapeutics have varying serum half lives, and those peptides andproteins with a short serum half-life, although having a promisingtherapeutic potential, are thus often unsuitable for further drugdevelopment. For example, albumin and gamma immunoglobulins (IgGs) areknown to have very long serum half lives of up to 20 days [1]. Inaddition, the Fc domain of other IgGs can be engineered to alter theirbinding interactions to neonatal Fc receptor as a method of prolongingserum half-life [2]. However, many other natural human proteins, such asinsulin, antibody fragments such as antigen binding fragments (Fabs) orsingle chain variable fragment (scFvs), and short peptides usually havemuch shorter serum half-lives ranging from minutes to approximately only1 hour. An efficient, cost effective and safe way of extending serumhalf lives of proteins and peptides with short half-lives is thereforecritical for these molecules to become therapeutic drugs, diagnostictools, etc.

Several strategies have been developed in an effort to prolong the serumhalf-life of proteins and other peptidic molecules that are short-livedin serum to avoid clearance of such therapeutic or diagnostic proteinsfrom circulation. Several technologies employed to facilitate serumhalf-life extension of proteins and peptidic molecules includeconjugation with a chemical attachment such as polyethylene glycol (i.e.pegylation) [3], fusion to the Fc region of an antibody [4], and fusionto a protein naturally having a long serum half life, such as albumin[5]. Unfortunately, these technologies suffer from complicationsincluding complex manufacturing and characterization processes, lowexpression levels, and undesired functions of the generated molecules.

Another approach that has been more recently developed to extend theserum half-life of proteins and other peptidic molecules employs the useof antibody fragments against serum proteins. Albumin, due mainly to itshigh serum concentration and long serum half-life, has been the mostselected target for this purpose. An isolated domain antibody againsthuman albumin was shown to prolong the serum half-life of interferon(IFN)-α2b after the two molecules were fused at the genetic level andexpressed as a fusion protein [6, 7]. The serum half-life of the newlygenerated fusion protein molecule is not only longer than that ofIFN-α2b, but even longer than that of the fusion protein of albumin andIFN-α2b.

Heavy chain variable domains of camelid heavy chain antibodies (HCAbs),known as VHH or single domain antibodies (sdAbs), have also beenexploited for this purpose. For example, a sdAb against human albuminwas shown to extend the serum half-life of an anti-TNFα sdAb fragmentfrom less than one hour to over two days [8].

Another serum protein with a long half life is transferrin. Transferrinis a plasma glycoprotein that transfers iron ions and has a serumconcentration of approximately 3 g/L and serum half-life of 7-8 days.Thus, transferrin is an ideal fusion partner to extend the serum halflife of peptidic molecules with unsatisfactory pharmacokinetics. Studieshave shown that fusion to transferrin significantly extended the serumhalf-life of both glucagon-like peptide 1 (GLP1) [9] and acetylcholinereceptor [10].

However, the use of antibodies and antibody fragments such as sdAbsagainst transferrin, for increasing the serum half-life of peptidicmolecules has not been reported. New methods for increasing the serumhalf-life of proteins and for producing proteins with improved serumhalf-life, that are efficient, cost-effective, and produce such proteinsin high yield would facilitate the development of novel protein-baseddiagnostics or therapeutics. Embodiments of the present invention relateto such methods.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to novel antibodies against transferrin,and particularly single domain antibodies (sdAbs) against transferrin.The present invention also relates to methods of using antibodies thatspecifically bind transferrin to increase the half-life of a targetprotein in the presence of the transferrin, novel fusion proteinscomprising the target protein having an increased half-life in thepresence of the transferrin, and compositions comprising the antibodiesthat specifically bind transferrin, or a fusion protein according to theinvention.

In one general aspect, the present invention relates to an isolatedantibody or fragment thereof that specifically binds a transferrin, theantibody or fragment thereof comprising one or more selected from thegroup consisting of:

-   -   (a) a complementarity determining region (CDR) 1 comprising an        amino acid sequence selected from the group consisting of        GSGFGINGVI (SEQ ID NO: 1); GSGFGVNGVI (SEQ ID NO: 2); GNVFTIAAMG        (SEQ ID NO: 3); GNVFTIAAMA (SEQ ID NO: 4); GNVFTIDAMG (SEQ ID        NO: 5); GSVFSIDAMG (SEQ ID NO: 6); GNVFGIDAVG (SEQ ID NO: 7);        GSIFSIKVMG (SEQ ID NO: 8); and GSIFPLNDMG (SEQ ID NO: 9);

(b) a CDR2 comprising an amino acid sequence selected from the groupconsisting of LIKSDGYTNYRESVKG (SEQ ID NO: 10); LIKSDGYTNYRESVRG (SEQ IDNO: 11); GITTGGSTNYADSVKG (SEQ ID NO: 12); GMTNGGKTNYADSVKG (SEQ ID NO:13); AMTNAGSTNYADSVKG (SEQ ID NO: 14); ATTTSGSSTNYADSVKG (SEQ ID NO:15); DITSGGSTDYSDSVKG (SEQ ID NO: 16); and TITRGGTTNYADSVKG (SEQ ID NO:17); and

-   -   (c) a CDR3 comprising an amino acid sequence selected from the        group consisting of PGVP (SEQ ID NO: 18); VTKWAARVGGSAEYE (SEQ        ID NO: 19); RSKLIATINNPYDY (SEQ ID NO: 20); RSKLIARINNPYEY (SEQ        ID NO: 21); RPKQATLIRDDY (SEQ ID NO: 22); DLGCSGAGSCPDY (SEQ ID        NO: 23); and DNRVGGSY (SEQ ID NO: 24).

Preferably, the antibody or fragment thereof is a single domain antibody(sdAb). More preferably, the antibody or antibody fragment thereof is ansdAb comprising the amino acid sequence of

A60219: (SEQ ID NO: 26)QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGV PFGQGTQVTVSS; A60401:(SEQ ID NO: 28) QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEWGQGTQVTVSS; A69449: (SEQ ID NO: 56)QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEWGQGTQVTVSS; A69433: (SEQ ID NO: 58)QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARP KQATLIRDDWGQGTQVTVSS;or A69447: (SEQ ID NO: 62)QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDN RVGGSYWGQGTQVTVSS.

The present invention also relates to a fusion protein comprising anantibody or fragment thereof according to embodiments of the presentinvention, a target protein, and optionally a linker, wherein theantibody or fragment thereof is fused to the carboxyl-terminus oramino-terminus of the target protein, and the linker optionallyseparates the antibody and the carboxyl-terminus or amino-terminus ofthe target protein.

The present invention also relates to a nucleic acid molecule comprisinga cDNA or synthetic DNA encoding an antibody or fragment thereofaccording to embodiments of the present invention, or a nucleic acidencoding a fusion protein according to embodiments of the presentinvention, and related expression vectors and host cells.

In another general aspect, the present invention relates to a method forincreasing the half-life of a target protein. According to embodimentsof the invention, the method comprises:

-   -   (1) obtaining a fusion protein, wherein the fusion protein        comprises an antibody or fragment thereof that specifically        binds a transferrin, the target protein, and optionally a        linker, wherein the antibody or fragment thereof is fused to a        carboxyl-terminus or amino-terminus of the target protein, and        the linker optionally separates the antibody and the        carboxyl-terminus or amino-terminus of the target protein; and    -   (2) exposing the fusion protein to the transferrin to thereby        increase the half-life of the target protein in the fusion        protein compared to the target protein alone.

According to an embodiment of the present invention, the fusion proteinis obtained by a method comprising:

-   -   (a) obtaining an expression vector encoding the fusion protein;    -   (b) introducing the expression vector into a cell to obtain a        recombinant cell;    -   (c) growing the recombinant cell under conditions to allow        expression of the fusion protein; and    -   (d) obtaining the fusion protein from the recombinant cell.

Another general aspect of the invention relates to a compositioncomprising an effective amount of a fusion protein, wherein the fusionprotein comprises an antibody or fragment thereof that specificallybinds a transferrin, a target protein, and optionally a linker, whereinthe antibody or fragment thereof is fused to the carboxyl-terminus oramino-terminus of the target protein, and the linker optionallyseparates the antibody and the carboxyl-terminus or amino-terminus ofthe target protein. Preferably, the composition further comprises thetransferrin.

The present invention also relates to a method comprising exposing acomposition according to an embodiment of the present invention to thetransferrin to thereby increase the half-life of the target protein inthe fusion protein.

A further aspect of the present invention relates to a system forincreasing the half-life of a target protein, the system comprising:

-   -   (1) an expression vector comprising a first nucleotide sequence        encoding an antibody or fragment thereof that specifically binds        a transferrin, and optionally a second nucleotide sequence        encoding a linker, wherein the first and second nucleotide        sequences are operably linked,    -   (2) a host cell, and    -   (3) the transferrin.

Other aspects, features and advantages of the invention will be apparentfrom the following disclosure, including the detailed description of theinvention and its preferred embodiments and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the inventionthere are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown. In thedrawings:

FIG. 1 is a graph showing the immune response of llama against humantransferrin after being immunized with an antigen;

FIG. 2 is an alignment of the amino acid sequences of 19 isolated singledomain antibodies (sdAbs) that bind human transferrin; complementaritydetermining regions (CDRs) and framework regions (FRs) are separatedwith a space; the sequences are numbered according to Kabat numbering;the sdAb sequences shown include A60219 (SEQ ID NO: 26), A13152 (SEQ IDNO: 30), A12722 (SEQ ID NO: 34), A12680 (SEQ ID NO: 36), A12690 (SEQ IDNO: 38), A13154 (SEQ ID NO: 40), A13146 (SEQ ID NO: 42), A13149 (SEQ IDNO: 44), A12666 (SEQ ID NO: 46), A12659 (SEQ ID NO: 48), A13355 (SEQ IDNO: 50), A12692 (SEQ ID NO: 52), A60401 (SEQ ID NO: 28), A13376 (SEQ IDNO: 32), A69476 (SEQ ID NO: 54), A69449 (SEQ ID NO: 56), A69433 (SEQ IDNO: 58), A69441 (SEQ ID NO: 60), and A69447 (SEQ ID NO: 62);

FIGS. 3A and 3B are images of an SDS polyacrylamide gel of purifiedsdAbs A60219, A60401, A69433, A69447, and A69449, isolated fromEscherichia coli, the amino acid sequences of which are shown in FIG. 2;

FIGS. 4A and 4B show surface plasmon resonance (SPR) sensorgrams oftransferrin sdAbs binding to human transferrin; FIG. 4A shows SPRsensorgrams of sdAb A60401 binding to human transferrin at varyingconcentrations of sdAb of 2 nM, 1 nM, 0.5 nM, 0.25 nM, and 0.125 nM,from the top to the bottom of the plot; FIG. 4B shows SPR sensorgrams ofsdAb A60219 binding to human transferrin at varying concentrations ofsdAb A60401 of 2 nM, 1 nM, 0.5 nM, 0.25 nM, and 0.125 nM, from the topto the bottom of the plot;

FIG. 5 is a chromatogram of size exclusion chromatography (SEC) analysisof transferrin sdAbs A60401 and A60219;

FIGS. 6A, 6B, 6C, 6D, and 6E show graphs of thermo-denaturation curvesof transferrin sdAbs measured by circular dichroism (CD) at 202 nm, 208nm, and 217 nm; FIG. 6A shows thermo-denaturation curves of sdAbA602019; FIG. 6B shows thermo-denaturation curves of sdAb A60401; FIG.6C shows thermo-denaturation curves of sdAb A69433; FIG. 6D showsthermo-denaturation curves of sdAb A69447; and FIG. 6E showsthermo-denaturation curves of sdAb A60449; and

FIG. 7 shows the serum clearance of human transferrin alone, sdAb A60219alone, and sdAb A60219 after injection into a Wister rat supplementedwith human transferrin; a sample of blood was taken at the indicatedtimepoints and the concentrations of A60219 and human transferrin in theblood sample were determined by enzyme linked immunosorbent assay(ELISA) in three experimental groups.

DETAILED DESCRIPTION OF THE INVENTION

Various publications are cited or described in the background andthroughout the specification and each of these references is hereinincorporated by reference in its entirety. Discussion of documents,acts, materials, devices, articles or the like which has been includedin the present specification is for the purpose of providing context forthe present invention. Such discussion is not an admission that any orall of the matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. It must benoted that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural reference unless the context clearlydictates otherwise.

One of ordinary skill in the art will be familiar with the structure ofan antibody. The light and heavy chains each contain a variable regionthat is responsible for binding the target antigen. The variable regioncontains the antigen binding determinants of the molecule, thusdetermining the specificity of an antibody for its target antigen. Thevariable regions of the light and heavy chains comprise threecomplementarity determining regions (CDRs).

As used herein “complementarity determining region” or “CDR” refers toan amino acid sequence of a variable region of a heavy or light chain ofan antibody that contributes to specific recognition of, and bindingspecificity for, the antigen. The CDRs are referred to as CDR1, CDR2,and CDR3. According to embodiments of the present invention, at leastone of the sequences of CDR1, CDR2, and CDR3 contributes to specificrecognition of, and binding specificity for, an antibody or fragmentthereof against transferrin, and preferably against human transferrin.

An “antibody fragment” as used herein includes any suitableantigen-binding antibody fragment. For example, an antibody fragment cancomprise a single-chain variable region. According to embodiments of thepresent invention, an antibody is preferably a single-domain antibody(sdAb).

As used herein, “single-domain antibody” or “sdAb” refers to theantigen-binding site of a heavy-chain antibody (HCAb) of camelids andsharks, which is naturally devoid of light chains. Camelids includecamel, llama, and alpaca. The antigen-binding site of HCAb of camelidsis formed by a single variable domain designated VHH. The sdAbs usuallyexist as monomeric proteins having relatively small sizes. An sdAbaccording to the invention has three CDRs (CDR1, CDR2, and CDR3).

As used herein, “antibody or fragment thereof against transferrin,”“antibody or fragment thereof that specifically binds transferrin,” and“transferrin antibody,” shall all have the same meaning, and refer to anantibody or fragment thereof, that is capable of binding specifically totransferrin.

As used herein, “sdAb against transferrin,” “sdAb that specificallybinds transferrin,” and “transferrin sdAb,” shall all have the samemeaning, and refer to a single domain antibody that is capable ofbinding specifically to transferrin.

As used herein, “transferrin” broadly refers to a protein that bindsiron. Transferrin, as used in the present invention, can be atransferrin from any organism that produces transferrin, and is morepreferably a mammalian transferrin, such as human transferrin or monkeytransferrin, and is most preferably a human transferrin.

As used herein, “binds specifically to” or “against” when used inconnection with an antibody or fragment thereof and transferrin refersto the binding or interaction between the antibody and fragment thereof,such as an sdAb, and the transferrin. An antibody or fragment thereof,such as an sdAb, according to the invention binds to a transferrin witha dissociation constant (K_(D)) of between 10⁻⁶ and 10⁻⁹ M, or less, andpreferably with a dissociation constant of less than 10⁻⁹ M, e.g., adissociation constant in the nanomolar to picomolar range (10⁻⁹ to10⁻¹²).

Any method known in the art can be used for determining specificantigen-antibody binding including, for example, surface plasmonresonance (SPR), scatchard analysis and/or competitive binding assays,such as radioimmunoassay (RIA), enzyme immunoassays (EIA) and sandwichcompetition assays, and the different variants thereof known in the art,as well as other techniques mentioned herein. Methods for determiningthe binding affinities or dissociation constants are known to thoseskilled in the art.

Any method known in the art can be used for characterizing an antibodyor sdAb according to the invention, such as SDS-polyacrylamide gelelectrophoresis (PAGE), circular dichroism (CD), size exclusionchromatography (SEC), etc. Methods for characterizing proteins, i.e.determining the oligomeric state, melting temperature, molecular weight,purity, etc., are known to those skilled in the art.

As used herein, the “half-life” of a protein or polypeptide refers tothe time taken for the concentration of the polypeptide to be reduced by50% in an assay conducted in vivo or in vitro. The reduction can becaused by degradation, clearance, or sequestration of the polypeptide inthe assay. The half-life of a polypeptide can be determined by anymanner known in the art in view of the present disclosure, such as bypharmacokinetic analysis. For example, to measure the half-life of aprotein or polypeptide in vivo, a suitable dose of the polypeptide isadministered to a warm-blooded animal (i.e. to a human or to anothersuitable mammal, such as a mouse, rabbit, rat, pig, dog, or a primate);blood samples or other samples from the animal are collected; the levelor concentration of the protein or polypeptide in the sample isdetermined; and the time until the level or concentration of thepolypeptide has been reduced by 50% compared to the initial level upondosing is calculated based on measured data. See, e.g., Kenneth, A etal., Chemical Half-life of Pharmaceuticals: A Handbook for Pharmacistsand Peters et al., Pharmacokinetic analysis: A Practical Approach(1996).

As used herein, “an increase in half-life” or “longer half-life” refersto an increase in any one of the parameters used to describe the proteinhalf-life, such as the t½-α, t½β and the area under the curve (AUC), anytwo of these parameters, or essentially all of these parameters, ascompared to a control.

As used herein, a “fusion tag” is a polypeptide sequence that can beoperably linked to a target protein or polypeptide to generate a fusionprotein for the ease of subsequent manipulation, such as for theexpression, purification, in vitro and in vivo analysis andcharacterization of the protein, or diagnostic or therapeuticapplication. A fusion tag can exhibit one or more properties. Forexample, the fusion tag can selectively bind to a purification mediumthat contains a binding partner for the fusion tag and allows theoperably linked target protein or fusion protein to be easily purified.The fusion tag can be, for example, glutathione S-transferase (GST),maltose binding protein, polyhistidine (His-tag), FLAG-tag, avidin,biotin, streptavidin, chitin binding domain, a ligand of a cellularreceptor, the Fc region of an antibody, green fluorescent protein, etc.

The present invention relates to antibodies or fragments thereof againsttransferrin and methods of using antibodies or fragments thereof againsta transferrin to increase the half-life of a target protein, i.e., byobtaining a fusion protein comprising a target protein and an antibodyor fragment thereof against a transferrin, and exposing the fusionprotein to the transferrin, for example, in a serum. The fusion proteincan be exposed to the transferrin in a serum either in vitro or in vivo.In a particular embodiment, the invention relates to novel sdAbs againsttransferrin and their uses.

Accordingly, in one general aspect, the present invention provides anisolated antibody or fragment thereof that specifically binds atransferrin, the antibody or fragment thereof comprising one or moreselected from the group consisting of:

-   -   (a) a complementarity determining region (CDR) 1 comprising an        amino acid sequence selected from the group consisting of        GSGFGINGVI (SEQ ID NO: 1); GSGFGVNGVI (SEQ ID NO: 2); GNVFTIAAMG        (SEQ ID NO: 3); GNVFTIAAMA (SEQ ID NO: 4); GNVFTIDAMG (SEQ ID        NO: 5); GSVFSIDAMG (SEQ ID NO: 6); GNVFGIDAVG (SEQ ID NO: 7);        GSIFSIKVMG (SEQ ID NO: 8); and GSIFPLNDMG (SEQ ID NO: 9);    -   (b) a CDR2 comprising an amino acid sequence selected from the        group consisting of LIKSDGYTNYRESVKG (SEQ ID NO: 10);        LIKSDGYTNYRESVRG (SEQ ID NO: 11); GITTGGSTNYADSVKG (SEQ ID NO:        12); GMTNGGKTNYADSVKG (SEQ ID NO: 13); AMTNAGSTNYADSVKG (SEQ ID        NO: 14); ATTTSGSSTNYADSVKG (SEQ ID NO: 15); DITSGGSTDYSDSVKG        (SEQ ID NO: 16); and TITRGGTTNYADSVKG (SEQ ID NO: 17); and    -   (c) a CDR3 having comprising an amino acid sequence selected        from the group consisting of PGVP (SEQ ID NO: 18);        VTKWAARVGGSAEYE (SEQ ID NO: 19); RSKLIATINNPYDY (SEQ ID NO: 20);        RSKLIARINNPYEY (SEQ ID NO: 21); RPKQATLIRDDY (SEQ ID NO: 22);        DLGCSGAGSCPDY (SEQ ID NO: 23); and DNRVGGSY (SEQ ID NO: 24).

According to embodiments of the present invention, an isolated antibody,preferably an sdAb, or fragment thereof, comprises a CDR 1 selected fromthe group consisting of SEQ ID NOs: 1-9, a CDR 2 selected from the groupconsisting of SEQ ID NOs: 10-17, and a CDR 3 selected from the groupconsisting of SEQ ID NOs: 18-24.

According to embodiments of the present invention, an isolated antibodyor fragment thereof that specifically binds a transferrin has anaffinity (K_(D)) for transferrin that is between 10⁻⁶ and 10⁻⁹M or less,and preferably has an affinity lower than 10⁻⁹ M. In a most preferredembodiment, an isolated antibody or fragment thereof according to theinvention has a K_(D) for transferrin that is in the subnanomolar range,for example, in the picomolar range, such as 1-10 pM, 15 pM, 20 pM, 30pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM.

In a particular embodiment, an isolated antibody or fragment thereofaccording to an embodiment of the present invention comprises a CDR1amino acid sequence of SEQ ID NO: 1, a CDR2 amino acid sequence of SEQID NO: 10, and a CDR3 amino acid sequence of SEQ ID NO: 18. In yetanother particular embodiment, an isolated antibody or fragment thereofaccording to the present invention comprises a CDR1 amino acid sequenceof SEQ ID NO: 3, a CDR2 amino acid sequence of SEQ ID NO: 12, and a CDR3amino acid sequence of SEQ ID NO: 19. In other particular embodiments,an isolated antibody or fragment thereof according to the presentinvention comprises a CDR1 amino acid sequence of SEQ ID NO: 6, a CDR2amino acid sequence of SEQ ID NO: 14, and a CDR3 amino acid sequence ofSEQ ID NO: 21; a CDR1 amino acid sequence of SEQ ID NO: 7, a CDR2 aminoacid sequence of SEQ ID NO: 15, and a CDR3 amino acid sequence of SEQ IDNO: 22; or a CDR1 amino acid sequence of SEQ ID NO: 9, a CDR2 amino acidsequence of SEQ ID NO: 17, and a CDR3 amino acid sequence of SEQ ID NO:24.

According to embodiments of the present invention, an antibody orfragment thereof, such as an sdAb, that specifically binds a transferrincan comprise an amino acid sequence selected from the group consistingof SEQ ID NOs: 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, and 62.

According to a preferred embodiment of the present invention, anantibody or fragment thereof that specifically binds transferrin is ansdAb. For example, an sdAb according to the invention that specificallybinds transferrin can be a camelid V_(H)H antibody.

In a particularly preferred embodiment of the present invention, an sdAbthat specifically binds transferrin comprises the

amino acid sequence of A60219: (SEQ ID NO: 26)QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGV PFGQGTQVTVSS;the amino acid sequence of A60401: (SEQ ID NO: 28)QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEYWGQGTQVTVSS; the amino acid sequence of A69449:(SEQ ID NO: 56) QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEYWGQGTQVTVSS; the amino acid sequence of A69433:(SEQ ID NO: 58) QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARPKQATLIRDDYWGQGTQVTVSS; or the amino acid sequence of A69447:(SEQ ID NO: 62) QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDN RVGGSYWGQGTQVTVSS.

The present invention also provides a nucleic acid comprising acomplementary DNA (cDNA) sequence encoding an antibody or fragmentthereof according to an embodiment of the invention. Also provided arevectors comprising the nucleic acid molecule, particularly expressionvectors, and recombinant host cells comprising the vectors that cansubsequently be used for downstream applications such as expression,purification, etc. The nucleic acid molecules, vectors and host cellscan be obtained using methods known in the art in view of the presentdisclosure.

An antibody or fragment thereof according to embodiments of theinvention can be produced recombinantly from a recombinant host cellusing methods known in the art in view of the present disclosure. Therecombinantly produced antibody or fragment thereof can be differentfrom the naturally occurring antibody or fragment thereof, for example,in posttranslational modification of amino acids. As used herein,“posttranslational modification of amino acids” refers to anymodification to the amino acids after translation of the amino acids,such as by attaching to one or more amino acids independently one ormore biochemical functional groups (such as acetate, phosphate, variouslipids and carbohydrates), changing the chemical nature of an amino acid(e.g. citrullination), or making structural changes (e.g. formation ofdisulfide bridges).

According to embodiments of the present invention, a nucleic acidmolecule comprises a cDNA or synthetic DNA sequence encoding an aminoacid sequence selected from the group consisting of SEQ ID NOs: 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, and 62.In particular embodiments of the present invention, the nucleic acidmolecule comprises a cDNA or synthetic DNA sequence comprising asequence selected from the group consisting of SEQ ID NOs: 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59 and 61.However, one skilled in the art will recognize that the specificnucleotide sequences listed are intended to be non-limiting examples,and various nucleotide sequences can encode the same amino acidsequence.

As used herein, a “cDNA or synthetic DNA” refers to a DNA molecule thatis different from a naturally occurring DNA molecule in at least one ofthe nucleotide sequence and the physical or chemical property of the DNAmolecule. For example, the “cDNA or synthetic DNA” can be different fromthe naturally occurring DNA in nucleotide sequence by not containing oneor more introns present in the natural genomic DNA sequence. The “cDNAor synthetic DNA” can also be different from a naturally occurring DNAin one or more physical or chemical properties, such as having adifferent DNA modification, regardless of whether the “cDNA or syntheticDNA” comprises the same or different nucleotide sequence as that of thenaturally occurring DNA.

As used herein, “DNA modification” refers to any modification to theDNA, such as by independently attaching to one or more nucleotides ofthe DNA one or more biochemical functional groups (such as a methylgroup, phosphate group, etc). Different host cells can have differentDNA modification systems, thus producing different DNA molecules eventhough the DNA molecules can have identical nucleotide sequence.

A “cDNA or synthetic DNA” can be made by any method in vivo or in vitroso long as the obtained “cDNA or synthetic DNA” is distinguishable froma naturally occurring DNA molecule. For example, a “cDNA” can be madefrom a messenger RNA (mRNA) template in a reaction catalyzed by theenzymes reverse transcriptase and DNA polymerase, or RNA-dependent DNApolymerase. In one embodiment, a “cDNA” can be made and amplified via areverse transcriptase polymerase chain reaction (RT-PCR) with thedesired mRNA template and DNA primers. A “synthetic DNA” can be made invitro using any method known in the art. A “synthetic DNA” can also bemade in vivo in a host cell that does not naturally contain a nucleicacid molecule having the identical nucleotide sequence as that of the“synthetic DNA,” such that the “synthetic DNA” made by the host cell isdistinguishable from any naturally occurring DNA sequence in at leastone or more physical or chemical properties, such as DNA methylation.

Embodiments of the present invention also relate to methods ofrecombinantly expressing and purifying an antibody or fragment thereofthat specifically binds transferrin. According to embodiments of thepresent invention, the method comprises obtaining an expression vectorencoding an antibody or fragment thereof according to an embodiment ofthe invention, introducing the expression vector into a host cell toobtain a recombinant cell, growing the recombinant cell under conditionsthat allow expression of the antibody or fragment thereof, and obtainingthe antibody or fragment thereof from the recombinant cell. The antibodyor fragment thereof that specifically binds transferrin can be isolatedby applying the lysate, supernatant, or periplasmic extract of therecombinant cell comprising the antibody or fragment thereof, to anaffinity column associated with the transferrin.

In another embodiment, the antibody or fragment thereof thatspecifically binds a transferrin further comprises a fusion tag thatfacilitates purification of the antibody or fragment thereof from therecombinant cell, by for example, applying the lysate, supernatant, orperiplasmic extract of the recombinant cell comprising of the antibodyor fragment thereof, to an affinity column associated with a bindingpartner of the fusion tag. As an illustrative and non-limiting example,a fusion tag can be a 6×-HIS tag, and the 6×-HIS tagged antibody orfragment thereof can be purified from the recombinant cell by applyingthe lysate, supernatant, or periplasmic extract of the recombinant cellto a nickel column.

Antibodies or fragments thereof, and particularly sdAbs, according toembodiments of the invention that specifically bind transferrin havehigh affinity for transferrin (e.g., picomolar range) and a longerhalf-life in a serum supplemented with transferrin, as compared to thehalf-life in a serum that is not supplemented with the transferrin.Without wishing to be bound by theory, it is believed that the bindinginteraction between the antibody and fragment thereof, such as sdAb, andthe transferrin contributes to the longer half-life of the antibody orfragment thereof in a serum. Such antibody or fragment thereof can thusbe used to increase the half-life of a target protein fused to theantibody or fragment thereof in a serum, or in a composition comprisingthe transferrin.

Thus, in another general aspect, the present invention relates to amethod of increasing the half-life of a target protein in a serum. Themethod comprises (1) obtaining a fusion protein, wherein the fusionprotein comprises an antibody or fragment thereof that specificallybinds a transferrin, the target protein, and optionally a linker,wherein the antibody or fragment thereof is fused to thecarboxyl-terminus or amino-terminus of the target protein, and thelinker optionally separates the antibody and the carboxyl-terminus oramino-terminus of the target protein; and (2) exposing the fusionprotein to the transferrin to thereby increase the half-life of thetarget protein in the fusion protein compared to the target proteinalone.

According to embodiments of the present invention, the transferrin usedin the exposing step can be present in any composition, including aserum or a buffered composition made in vitro. Preferably, the fusionprotein is exposed to the transferrin by administering it to a serumcomprising the transferrin.

According to embodiments of the present invention, when exposed to thetransferrin, the fusion protein has an increased half-life, asdetermined, for example by measuring the half-life, as compared to thetarget protein alone. The fusion protein can optionally contain a linkerthat fuses the target protein to the antibody or fragment thereof, andalso functions to separate the target protein from the antibody orfragment thereof. Linkers that can be used to fuse two protein moleculestogether will be well known to those skilled in the art in view of thepresent disclosure.

The antibody or fragment thereof can be fused to the target protein byany method known in the art in view of the present disclosure, such as,for example, via genetic fusion or covalent linkage. The antibody orfragment thereof can be fused to either the amino-terminus or thecarboxyl-terminus of the target protein.

Preferably, the fusion protein comprises an antibody or fragment thereofaccording to embodiments of the present invention. For example, theantibody or fragment thereof comprises an amino acid sequence selectedfrom the group consisting of the amino acid sequence of CDR1 comprisingthe sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9; the amino acidsequence of CDR2 comprising the sequence of SEQ ID NO: 10, 11, 12, 13,14, 15, 16, or 17; and the amino acid sequence of CDR3 comprising thesequence of SEQ ID NO: 18, 19, 20, 21, 22, 23, or 24. Most preferably,the antibody or fragment thereof of the fusion protein is an sdAb andeven more preferably is an sdAb comprising the amino acid sequence ofA60219 (SEQ ID NO: 26), A60401 (SEQ ID NO: 28), A69449 (SEQ ID NO: 56),A69433 (SEQ ID NO: 58), or A69447 (SEQ ID NO: 62).

According to embodiments of the present invention, the target protein isa peptide or polypeptide, such as a therapeutic polypeptide, apolypeptide that can be used for a diagnostic purpose, or a polypeptidefor structural-activity studies. For example, the target protein can bean antibody, peptide, or any other polypeptide that has been or will bedeveloped or used for a therapeutic or diagnostic purpose, or a proteinsubjected to structural and/or functional analysis. Preferably, thetarget protein is a therapeutic peptide or polypeptide that is unstablein serum, and in need of increased serum half-life to be used fortherapeutic, diagnostic purposes, etc.

The fusion proteins according to embodiments of the present inventioncan be used for various purposes using methods known in the art in viewof the present disclosure. For example, a fusion protein can be used forassaying the affinity of the target protein to a binding partner, e.g.,for drug screening or target identification purposes. It can also beused in a diagnostic method, particularly if the method involvesadministering the target protein to the serum. It can further be usedfor therapeutic purposes, particularly if the target protein is known tobe unstable in serum.

Another general aspect of the invention relates to a compositioncomprising an effective amount of a fusion protein, wherein the fusionprotein comprises an antibody or fragment thereof that specificallybinds a transferrin, a target protein, and optionally a linker, whereinthe antibody or fragment thereof is fused to the carboxyl-terminus oramino-terminus of the target protein, and the linker optionallyseparates the antibody and the carboxyl-terminus or amino-terminus ofthe target protein. Preferably, the composition further comprises thetransferrin, which increases the half-life of the fusion protein in thecomposition. The composition can further comprise a pharmaceuticallyacceptable carrier, which can comprise any carrier that is suitable forpharmaceutical or diagnostic purposes.

Depending on the use, the effective amount can be the amount of thefusion protein that is effective to provide a therapeutic or diagnosticuse of the target protein as part of the fusion. For example, aneffective amount of the fusion protein, when used for a therapeuticapplication, can be the amount fusion protein that treats and/orprevents a disease, disorder, or condition treatable or preventable bythe target protein as part of the fusion protein. An effective amount ofthe fusion protein, when used for a diagnostic application, can be theamount of the fusion protein needed to detect a marker, e.g.,biomolecule such as nucleic acid or protein, by the target protein aspart of the fusion protein.

A composition according to an embodiment of the present invention can beused in vivo or in vitro for any purpose. Compositions of the inventionfor in vivo uses can be formulated for any method of delivery to asubject, e.g., a mammal, such as human, rat, mice, monkey, or rabbit,including, but not limited to oral, topical, and injection. Preferably acomposition for in vivo use is formulated for injection or intravenousadministration.

The present invention also relates to a method comprising exposing acomposition according to an embodiment of the present invention to thetransferrin to thereby increase the half-life of the target protein inthe fusion protein.

In one embodiment, the present invention relates to a method comprisingexposing a composition according to an embodiment of the presentinvention to the transferrin used in the fusion protein, for example, byadministering the composition to a serum comprising transferrin, in vivoor in vitro for identifying a diagnostic or therapeutic agent.

In another embodiment, a method according to the present inventioncomprises administering a composition according to an embodiment of thepresent invention to a subject in need of treatment by the targetprotein, wherein the composition comprises a therapeutically effectiveamount of a fusion protein comprising an antibody or fragment thereofthat specifically binds the human transferrin and a target protein.

In yet another embodiment, a method according to the invention comprisesadministering the composition to a subject in need of a diagnosis by thetarget protein, wherein the composition comprises a diagnosticallyeffective amount of the fusion protein.

Embodiments of the present invention also relate to compositionscomprising a fusion protein according to the invention, and methods forincreasing the half-life of a target protein in a composition. A methodfor increasing the half-life of a target protein in a compositioncomprises obtaining a fusion protein, preferably isolated fusionprotein, comprising an antibody or fragment thereof against atransferrin, the target protein and an optional linker, wherein theantibody or fragment thereof is fused to the carboxyl-terminus oramino-terminus of the target polypeptide, and the optional linkerseparates the antibody or fragment thereof and the carboxyl-terminus oramino-terminus of the target polypeptide; and exposing the fusionprotein to the transferrin in the composition, wherein the fusionprotein has a longer half-life than the target protein alone in thecomposition.

Without wishing to be bound by theory, it is believed that the specificbinding between the antibody or fragment thereof in the fusion proteinand the transferrin in a composition or a serum contributes to increasedhalf-life of the target protein.

Embodiments of the present invention also provides methods for obtaininga target protein having increased serum half-life, and for expressingand purifying a fusion protein comprising an antibody or fragmentthereof that binds to a transferrin and a target protein.

According to embodiments of the present invention, a method forobtaining a target protein having increased serum half-life comprises:

-   -   (a) obtaining an expression vector encoding a fusion protein        comprising an antibody or fragment thereof that specifically        binds a transferrin, the target protein, and an optional linker,        wherein the antibody or fragment thereof is fused to the        carboxyl-terminus or amino-terminus of the target protein, and        the optional linker separates the antibody and the        carboxyl-terminus or amino-terminus of the target protein;    -   (b) introducing the expression vector of step (a) into a cell to        obtain a recombinant cell;    -   (c) growing the recombinant cell under conditions to allow        expression of the fusion protein; and    -   (d) obtaining the fusion protein from the recombinant cell.

Expression vectors encoding the fusion protein and recombinant cellsexpressing the fusion protein can be constructed using methods known inthe art in view of the present disclosure. Any host cell suitable forrecombinant production of the fusion protein can be used such as amammalian cell, plant cell, yeast cell, or bacterial cell. Preferably,the host cell is a bacterial cell, and is more preferably Escherichiacoli. Any method for obtaining the fusion protein from the recombinantcell can be used in view of the present disclosure including, but notlimited to, column chromatography such as affinity chromatography.

In one embodiment, the fusion protein can be obtained from a recombinantcell and purified by utilizing the specific interaction between theportion of the fusion protein comprising the antibody or fragmentthereof that specifically binds transferrin, and transferrin, to obtainthe fusion protein from the recombinant cell, e.g., by affinitychromatography using an affinity column associated with transferrin.

In another embodiment, the fusion protein can further comprise a fusiontag at the amino-terminus or carboxyl-terminus of the fusion protein tofacilitate obtaining and purifying the fusion protein from therecombinant cell. The fusion tag can be fused to the transferrin, or tothe antibody or fragment thereof that binds transferrin. For example,the lysate, periplasmic extract, or supernatant of the recombinant cellcomprising the fusion protein can be obtained and applied to a columnassociated with the appropriate binding partner of the fusion tag. In aparticular and non-limiting example, the fusion protein can furthercomprise a His-tag and the lysate, periplasmic extract, or supernatantof the recombinant cell comprising the fusion protein can be obtainedand applied to a nickel column to obtain the fusion protein from therecombinant cell. The column can then be washed, and the fusion proteineluted from the column under the appropriate buffering conditions toobtain the fusion protein.

Another general aspect of the present invention relates to a system forincreasing the half-life of a target protein, comprising:

-   -   (1) an expression vector comprising a first nucleotide sequence        encoding an antibody or fragment thereof that specifically binds        a transferrin, and optionally a second nucleotide sequence        encoding a linker, wherein the first and second nucleotide        sequences are operably linked;    -   (2) a host cell; and    -   (3) the transferrin.

The expression vector can be used to construct an expression vector fora fusion protein comprising an antibody or fragment thereof thatspecifically binds a transferrin, a target protein, and optionally alinker, wherein the antibody or fragment thereof is fused to thecarboxyl-terminus or amino-terminus of the target protein, and thelinker optionally separates the antibody and the carboxyl-terminus oramino-terminus of the target protein. Thus, in certain embodiments ofthe invention, an expression vector comprises a first nucleotidesequence encoding an antibody or fragment thereof that specificallybinds a transferrin, optionally a second nucleotide sequence encoding alinker, and a third nucleotide sequence encoding a target protein,wherein the first, second, and third nucleotide sequences are operablylinked, such that the antibody or fragment thereof is fused to thecarboxyl-terminus or amino-terminus of the target protein, and thelinker optionally separates the antibody and the carboxyl-terminus oramino-terminus of the target protein.

The host cell can be used to construct a recombinant cell for expressingthe fusion protein, e.g., by transforming the host cell with theexpression vector for the fusion protein, using any method known in theart in view of the present invention, including but not limited toelectroporation and calcium chloride transformation.

The transferrin can be used to stabilize the fusion protein. It can alsobe used to isolate the fusion protein by affinity chromatography.

According to an embodiment of the present invention, the system canfurther comprise a solid support for capturing the fusion protein viaspecific binding between the antibody or fragment thereof in the fusionprotein and the transferrin associated with the solid support, or viaspecific binding between a fusion tag on the fusion protein and abinding partner of the fusion tag associated with the solid support.

The system can further comprise one or more buffers useful for theexpression and/or isolation of the fusion protein.

The following specific examples of the invention are furtherillustrative of the nature of the invention, and it needs to beunderstood that the invention is not limited thereto.

EXAMPLES Example 1: Production and Characterization of sdAbs thatSpecifically Bind a Transferrin

Materials and Methods

Isolation of Transferrin sdAbs from a Llama Immune Phage Display Library

A male llama (Lama glama) was injected subcutaneously with 100 μg, 50μg, 50 μg, 10 μg, and 10 μg human transferrin on days 1, 22, 36, 50 and64, respectively [11]. Complete Freund's Adjuvant (Sigma, St. Louis,Mo.) was used for the primary immunization and Incomplete Freund'sAdjuvant was used for subsequent immunizations 2-4. Adjuvant was notused for the final immunization. The llama was bled one week followingeach immunization and heparinized blood was collected for immediateisolation of the peripheral blood leukocytes, which were then stored at−80° C. until further use.

Total RNA was isolated from 1×10⁸ leukocytes using a QIAamp RNA BloodMini Kit (Qiagen). cDNA was synthesized using pd(N)₆ as primer and 566ng total RNA as the template. Four forward primers P441_VHHF1(GCCCAGCCGGCCATGGCCSMBGTRCAGCTGGTGGAKTCTGGGGGA; SEQ ID NO: 63),P442_VHHF2 (GCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA; SEQ ID NO:64), P759_VHHF3 (GCCCAGCCGGCCATGGCCCAGGTACAGCTGGTGGAGTCT; SEQ ID NO: 65)and P444_VHHF4 (GCCCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTGTGG; SEQ ID NO:66); and two reverse primers P445_CH2R (CGCCATCAAGGTACCAGTTGA; SEQ IDNO: 67) and P446_CH2b3R (GGGGTACCTGTCATCCACGGACCAGCTGA SEQ ID NO: 68)were used to amplify V_(H)-C_(H)1-Hinge-C_(H)2 region of conventionalimmunoglobulin G antibody (IgG) or V_(H)H-Hinge-C_(H)2 of heavy chainantibody. Amplified V_(H)H products of approximately 600 bp from theprimer combinations of P445_C_(H)2R with each of the forward primersP441_VHHF1, P442_VHHF2, P759_VHHF3, and P444_VHHF4 were extracted from a1% agarose gel, and purified with a QIAquick Gel Extraction Kit (Qiagen)and the amplified products from primers P446_C_(H)2R were PCR purified.In a second PCR reaction, two primers, P440_VHHF(CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC; SEQ ID NO: 69) and P447_VHHR(CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTG; SEQ ID NO: 70) wereused to introduce SfiI restriction sites, and to amplify the final sdAbfragments from the combined amplified products. The final PCR productwas digested with SfiI and ligated into a conventional phagemid vectorconstructed at GenScript Inc., and transformed into E. coli TG1 byelectroporation. Phage were rescued and amplified with helper phageM13KO7 (New England Biolabs (NEB)).

The llama immune phage display library was panned against transferrinthat was conjugated to M-280 beads (Invitrogen). Approximately 3×10¹¹phages were added to the beads and incubated at 37° C. for 2 hours forantigen binding. After disposal of unbound phages, the beads were washedsix times with phosphate buffered saline supplemented with 0.05% Tween20 (PB ST) for round one, and the washes were increased by one for eachadditional round. Phages were eluted by 10 min incubation with 100 μl100 mM triethylamine, and the eluate was subsequently neutralized with200 μl M Tris-HCl (pH 7.5). Phages were amplified as described above,but on a smaller scale. After two rounds of panning, eluted phages wereused to infect exponentially growing E. coli TG1. Individual colonieswere used in phage enzyme-linked immunosorbent assay (ELISA).

For phage ELISA, a 96-well microliter plate was coated overnight with 2μg/ml human transferrin and then blocked with 4% modified phosphatebuffered saline (MPBS) for 2 hours at 37° C. Phage from individualclones were pre-blocked with 4% MPBS overnight, added to the pre-blockedwells and incubated for 1 hour. Phage ELISA was performed using the GEHealthcare Detection Module Recombinant Phage Antibody System (GEHealthcare, Uppsala, Sweden) and positive phage clones were sequenced.

Expression of sdAbs

DNA encoding each sdAb (A60401, A60219, A69433, A69447, or A69449) (FIG.2) was cloned into the BbsI and BamHI sites of a periplasmic expressionvector pSJF2H [12], which added a 5× Histidine purification tag at theC-terminus of the sdAbs. These sdAbs were expressed periplasmically andpurified by immobilized metal ion affinity chromatography (IMAC) [13].Briefly, clones were inoculated in 25 ml LB-Ampicillin (Amp) andincubated at 37° C. with 200 rpm shaking overnight. The next day, 20 mlof the culture were used to inoculate 1 L of M9 medium (0.2% glucose,0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.1% NH₄Cl, 0.05% NaCl, 1 mM MgCl₂, 0.1 mMCaCl₂)) supplemented with 0.4% casamino acids, 5 mg/L of vitamin B1, and200 μg/ml of Amp, and cultured for 24 hours. 100 ml of 10×TB nutrients(12% Tryptone, 24% yeast extract and 4% glycerol), 2 ml of 100 mg/mlAmp, and 1 ml of 1 M isopropyl-beta-D-Thiogalactopyranoside (IPTG) wereadded to the culture and incubation was continued for another 65-70hours at 28° C. with 200 rpm shaking. E. coli cells were harvested bycentrifugation and lysed with lysozyme. Cell lysates were centrifuged,and clear supernatant was loaded onto High-Trap™ chelating affinitycolumns (GE Healthcare), and His-tagged proteins were purified.

Surface Plasmon Resonance (SPR) Analysis

Experiments were performed using a BIAcore T200 optical sensor platformand research grade CM5 sensor chips (GE Healthcare). Human transferrinwas immobilized on the sensor chip surface by standard amine coupling.All experiments were carried out in HEPES buffer [10 mM HEPES (pH 7.4),150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20] at 25° C. Antibodies wereinjected at serial dilutions ranging from 0.25 nM to 16 nM at a flowrate of 30 μl/min unless otherwise indicated. The amount of boundanalyte after subtraction from the blank control surface is shown asrelative response units (RU). The double referenced sensorgrams fromeach injection series were analyzed for binding kinetics using BIAevaluation software (GE Healthcare). Dissociation constants (K_(D)s)were calculated from the on- and off-rates (k_(on) and k_(off),respectively), as determined by global fitting of the experimental datato a 1:1 Langmuir binding model (Chi²<1).

Size Exclusion Chromatography

Size exclusion chromatography (SEC) analyses of sdAbs, A60401 andA60219, (FIG. 5) were performed with a Superdex 200™ column (GEHealthcare). Superdex separations were carried out in PBS. Low molecularweight (MW) markers ribonuclease A (13.7 kDa), chymotrypsin A (25 kDa)and ovalbumin (43 kDa) were used to calculate the MW of the sdAbs.

Measurement of Melting Temperature of sdAbs by Circular Dichroism

Proteins were separated with a Superdex75 SEC column in 10 mM phosphatebuffer, pH 7.0. Peaks representing major components of proteins werecollected and used in circular dichroism (CD) analysis. CD spectra werecollected from 250 to 200 nm at protein concentrations of 100 ug/ml in a10 mm quartz cuvette with a J-815 CD spectrometer (JASCO). CD spectrawere measured at 2° C. intervals from 30° C. to 90° C. to determinethermal denaturation of proteins with a temperature shift speed of 1°C./min. Ellipticity at 202 nm, 208 nm, and 217 nm was plotted againsttemperature, and melting temperatures (T_(m)s) were calculated as theaverage T_(m) at the three wavelengths.

Measurement of Serum Half-Life

A group of three female Wister rats, each weighing approximately 250 g,were intravenously (i.v.) injected with 30 mg human transferrin, and 500μg sdAb A60219, A60401, A69433, A69447, or A69449, respectively,immediately after injecting 30 mg human transferrin into the tail vein.Blood was collected from the eye through a glass capillary at indicatedtime points. Sera were separated and stored at −80° C. until furtheruse. Concentrations of the injected antibody molecules in the abovecollected samples were measured by ELISA.

For the detection of transferrin, anti-transferrin antibody [HTF-14](Abcam, ab769) was coated on microtitre plates (Costar, 9018) overnightat 4° C. at a concentration of 1 μg/ml. After washing three times withPBST, plates were blocked with 1% BSA in PBST for two hours at 37° C.Diluted sera (1% BSA in 0.05% PBS-T used as diluent) were added to thewells and incubated at 37° C. for 2 hours. After washing four times withPBST, HRP labeled anti-transferrin antibody (0.1 μg/ml) (Abcam, ab9538)was added to the wells and incubated for another 1 hour. After washingthe plate with PBST, the color was developed with TMB substrate for 10minutes, and the reaction was stopped by adding 1 M HCl. The absorbanceof each well was measured at 450 nm using a spectrometer. Serialdilutions of pure human transferrin in 1% BSA in PBST were used togenerate a standard curve for human transferrin concentration analysis.

The same method was used for the detection of sdAbs, except anti-sdAbrabbit polyclonal antibody (GenScript) was used as a capture antibody,and HRP labeled anti-sdAb rabbit polyclonal antibody (0.1 μg/ml)(GenScript) was used as detection antibody. Serial dilutions of puresdAbs in 1% BSA in PBST were used to make a standard curve for theconcentration of the sdAbs.

Results

Isolation and Characterization of sdAbs

Isolation of transferrin-specific sdAbs was achieved by llamaimmunization with human transferrin, construction of an immune phagedisplay library from the llama, and subsequent panning.

Human transferrin induced a medium immune response in the llama. Anapproximately 25,000 fold dilution of the serum after the fifthimmunization still detected positive (FIG. 1). This level of response isin agreement with what is usually achieved with llama immunization.

Approximately 1×10⁸ llama leukocytes were used for the isolation ofmRNA, which was then used for the construction of a phage library. Thesize of the obtained library was 2×10⁸ independent transformants with apositive insertion rate of 92%. Two rounds of phage display panning wereperformed on immobilized transferrin, and phage enrichment was observedduring panning (data not shown). Phage ELISA showed that approximately88% of the analyzed clones bound to transferrin. Analysis of encodingsequences of the sdAbs displayed on the phage clones revealed 19different sdAb amino acid sequences (SEQ ID NOs: 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 and 62, and FIG. 2).

Five sdAbs, A60219, A60401, A69433, A69447 and A69449, were sub-cloned,respectively, into an E. coli periplasmic expression vector, pSJF2H[12]. The sdAbs, tagged with a 6× histidine (His) tag at theirC-terminal ends, were then produced in E. coli and purified by IMAC(FIGS. 3A and 3B). The sdAb yields of A60219, A60401, A69433, A69447 andA69449 were 8.4, 8.5, 12.5, 17.2, and 7.8 mg per liter of TG1 culture,respectively.

The five transferrin sdAbs, A60219, A60401, A69433, A69447 and A69449,were analyzed for binding to transferrin using an SPR-based biosensor(FIG. 4). Among them, A69433, A69447 and A69449, showed excellent crossbinding activities with human and monkey transferrins (Table 2).

The results are shown in Table 1 and Table 2 below, and in FIG. 4. Forexample, the on-rates of the sdAbs were 9.27×10⁵ and 5.14×10⁷ M⁻¹s⁻¹,and the off-rates 6.22×10⁻⁵ and 6.74×10⁻⁴ s⁻¹ for A60401 and A60219,respectively. The dissociation constants (K_(D)s) of the sdAbs werecalculated as 67.14 pM for A60401 (FIG. 4A) and 13.1 pM for A60219 (FIG.4B).

TABLE 1 Transferrin sdAbs (A60219 and A60401) binding to humantransferrin measured using SPR. Antibody ID Antigen k_(on) (1/Ms)k_(off) (1/s) K_(D) (M) A60219 Human transferrin 5.14E+07 6.74E−041.31E−11 A60401 Human transferrin 9.27E+05 6.22E−05 6.71E−11

TABLE 2 Transferrin sdAbs (A69433, A69447 and A69449) binding to humanand monkey transferrins measured using SPR. Antibody ID Antigen k_(on)(1/Ms) k_(off) (1/s) K_(D) (M) A69433 Human Transferrin 1.11E+056.42E−04 5.76E−09 A69447 2.03E+05 2.09E−04 1.03E−09 A69449 3.42E+056.95E−04 2.03E−09 A69433 Monkey Transferrin 2.47E+05 5.93E−04 2.40E−09A69447 3.04E+05 7.58E−05 2.50E−10 A69449 4.71E+05 5.29E−04 1.12E−09

Characterization of the sdAbs

Size exclusion chromatography (SEC) was employed to evaluate whether thetwo sdAbs A60401 and A60219 existed as monomers (FIG. 5). As with mostcamelid sdAbs, A60401 was a pure monomer, which was demonstrated as asingle peak at 14.28 ml of elution volume through size exclusionchromatography with a Superdex75 column, corresponding to a measured MWof 10.6 kDa, very close to its calculated MW of 14,347 Da. A60219 verylikely exists as pure monomer as well, as it has a single elution peakat 13.98 ml, representing an estimated MW of 11.2 kDa. However, itsslightly faster elution from the column than A60401 indicates thatdynamic transfer between monomeric and oligomeric forms of A60219 mayexist, as it eluted faster than A60401 even with a smaller calculated MW(13,170 Da).

CD profiles of A60219 and A60401 were measured to estimate theirsecondary structures and thermo half-life. Both proteins had a CDprofile that is typical for single domain antibodies (Data not shown).Thermo-induced protein denaturation was measured in the temperaturerange from 30 to 90° C. at 2° C. intervals. Plotting the CD values at202 nm, 208 nm, and 217 nm against temperature suggested a two phasedenaturation for both A60219 (FIG. 6A) and A60401 (FIG. 6B) with acalculated melting temperature (T_(m)) of 69.8° C. and 58.7° C.,respectively.

Serum Clearance of sdAb A60219

A60219 was selected to test its serum half-life based on its highaffinity, pure monomeric status, and high thermo half-life. A60219 wasfirst injected into rat either by itself or immediately after injectionof human transferrin into the rats. Human transferrin was also injectedto measure its serum half-life in rats, mimicking an environment ofexisting human transferrin in the blood. An ELISA system was employed tomeasure the concentrations of A60219 in rat blood taken at differenttime points after injection.

Blood clearance of human transferrin in Wister rats was firstinvestigated. After injecting 30 mg human transferrin into the rats,blood samples were collected for seven days. A gradual and steadydecline of transferrin concentration was observed. Thet_(1/2β was calculated as) 22 hours. This is significantly shorter thanthe serum half-life of human transferrin in human. Nevertheless,infusion of human transferrin in rats provides a model for us toestimate the serum half-life of A60219 in human. It is noteworthy thatthe anti-human transferrin antibody has no cross reactivity with rattransferrin, which makes the analysis of the serum clearance of the sdAbsimple.

The serum half-life of the sdAb A60219 was then investigated. As manyother sdAbs ever studied in animal models, A60219 was cleared from therat blood rapidly. At approximately 8 hours only trace amounts ofinjected A60219 could be detected in the rat blood.

A60219 half-life was significantly longer in rats supplemented withhuman transferrin. Its blood clearance rate is very similar to that ofhuman transferrin. The calculated serum half-life is also 22 hours. Thisindicates that, when injected into human, A60219 would have a similarserum half-life as human transferrin.

Results from the serum clearance study demonstrated that, exposing apolypeptide comprising an antibody or fragment thereof against atransferrin to the transferrin significantly increased the serumhalf-life of the polypeptide in vivo.

REFERENCES

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We claim:
 1. An isolated single-domain antibody (sdAb) orantigen-binding fragment thereof that specifically binds a transferrin,wherein the sdAb or antigen-binding fragment thereof comprises acomplementarity determining region 1 (CDR1), CDR2, and CDR3 having theamino acid sequences, respectively, of: (a) SEQ ID NO: 1, SEQ ID NO: 10,and SEQ ID NO: 18; (b) SEQ ID NO: 3, SEQ ID NO: 12, and SEQ ID NO: 19;(c) SEQ ID NO: 6, SEQ ID NO: 14, and SEQ ID NO: 21; (d) SEQ ID NO: 7,SEQ ID NO: 15, and SEQ ID NO: 22; or (e) SEQ ID NO: 9, SEQ ID NO: 17,and SEQ ID NO:
 24. 2. The isolated sdAb or antigen-binding fragmentthereof of claim 1, comprising the CDR1, CDR2, and CDR3 having the aminoacid sequences, respectively, of SEQ ID NO: 3, SEQ ID NO: 12, and SEQ IDNO:
 19. 3. The isolated sdAb or antigen-binding fragment thereof ofclaim 1, comprising the CDR1, CDR2, and CDR3 having the amino acidsequences, respectively, of SEQ ID NO: 6, SEQ ID NO: 14, and SEQ ID NO:21.
 4. The isolated sdAb or antigen-binding fragment thereof of claim 1,comprising the CDR1, CDR2, and CDR3 having the amino acid sequences,respectively, of SEQ ID NO: 7, SEQ ID NO: 15, and SEQ ID NO:
 22. 5. Theisolated sdAb antibody or antigen-binding fragment thereof of claim 1,comprising the CDR1, CDR2, and CDR3 having the amino acid sequences,respectively, of SEQ ID NO: 9, SEQ ID NO: 17, and SEQ ID NO:
 24. 6. Theisolated sdAb or antigen-binding fragment thereof of claim 1, comprisingthe amino acid sequence selected from the group consisting of: A60219:(SEQ ID NO: 26) QVQLVESGGGLVQAGGSLRLSCVASGSGFGINGVIWYRQAPGKQRELVALIKSDGYTNYRESVKGRFTISRDDAKNTVWLQMNALEPEDTGVYYCKTPGV PFGQGTQVTVSS; A60401:(SEQ ID NO: 28) QVKLEESGGGLVQAGGSLRLSCEASGNVFTIAAMGWFRQAPGKERELVAGITTGGSTNYADSVKGRFTISRDNAQNTMYLQMNSLRPEDTAAYSCNAVTKWAARVGGSAEYEYWGQGTQVTVSS; A69449: (SEQ ID NO: 56)QVQLVESGGGVVQAGGSLRLSCVASGSVFSIDAMGWYRQAPGNQRELVAAMTNAGSTNYADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCNARSKLIARINNPYEYWGQGTQVTVSS; A69433: (SEQ ID NO: 58)QVKLEESGGGLVQAGGSLRLSCVASGNVFGIDAVGWYRQAPGKQRELVAATTTSGSSTNYADSVKGRFTISRDIAKNTVYLQMDSLKPEDTAVYYCYARPKQATLIRDDYWGQGTQVTVSS; and A69447: (SEQ ID NO: 62)QVKLEESGGGSVQAGGSLRLSCTGSGSIFPLNDMGWYRQAPGKQRELVATITRGGTTNYADSVKGRFTISRDSNAKNTVYLQMNSLKVEDTAVYYCNMDN RVGGSYWGQGTQVTVSS.


7. The isolated sdAb antibody or antigen-binding fragment thereof ofclaim 1, having a dissociation constant (K_(d)) of 10⁻⁹ M or less forthe transferrin.
 8. The isolated sdAb antibody or antigen-bindingfragment thereof of claim 1, wherein the transferrin is a humantransferrin or monkey transferrin.
 9. The isolated sdAb antibody orantigen-binding fragment thereof of claim 1, being producedrecombinantly.
 10. An isolated single-domain antibody (sdAb) antibody orantigen-binding fragment thereof that specifically binds a transferrin,wherein the sdAb or antigen-binding fragment thereof comprises acomplementarity determining region 1 (CDR1), CDR2, and CDR3 having theamino acid sequences, respectively, of SEQ ID NO: 1, SEQ ID NO: 10, andSEQ ID NO:
 18. 11. The sdAb antibody or antigen-binding fragment thereofof claim 10, wherein the sdAb or antigen-binding fragment thereofcomprises the amino acid sequence of SEQ ID NO:
 26. 12. The sdAbantibody or antigen-binding fragment thereof of claim 10, having adissociation constant (K_(d)) of 10⁻⁹ M or less for the transferrin. 13.The sdAb antibody or antigen-binding fragment thereof of claim 11,having a dissociation constant (K_(d)) of 10⁻⁹ M or less for thetransferrin.
 14. The sdAb antibody or antigen-binding fragment thereofof claim 10, wherein the transferrin is a human transferrin or monkeytransferrin.
 15. The sdAb antibody or antigen-binding fragment thereofof claim 11, wherein the transferrin is a human transferrin or monkeytransferrin.
 16. The sdAb antibody or antigen-binding fragment thereofof claim 10, being produced recombinantly.
 17. The sdAb antibody orantigen-binding fragment thereof of claim 11, being producedrecombinantly.
 18. A nucleic acid comprising a cDNA or synthetic DNAencoding the isolated sdAb antibody or antigen-binding fragment thereofof claim
 1. 19. An expression vector comprising the nucleic acid ofclaim
 18. 20. A recombinant host cell comprising the expression vectorof claim 19.